PROJECT SUMMARY Failure in engraftment is one of the major limitations of hematopoietic stem cell transplantation, which is critical for the treatment of many benign and malignant hematologic disorders. Besides immune-mediated rejection of the transplanted cells, ineffective migration, adhesion, and engraftment of the hematopoietic stem and progenitor cells (HSPCs) to the bone marrow niche are important causes of engraftment failure. The intracellular signaling pathways regulating HSPC engraftment and migration to the bone marrow often directly or indirectly target the actin cytoskeleton network through proteins that regulate actin polymerization. The mDia formins are a major actin-nucleating family, but their functions in HSPCs are unknown. Our preliminary studies using our recently generated mDia2 hematopoietic-specific knockout mice showed that loss of mDia2 resulted in significant defects in HSPC adhesion and bone marrow engraftment in competitive transplantation assays. Loss of mDia2 also markedly reduced G-CSF-induced HSPC mobilization out of the bone marrow. We also found that beta2 integrins including CD11a/CD18 (LFA1) and CD11b/CD18 (Mac1) were significantly downregulated with the loss of mDia2. These results led us to hypothesize that mDia2 is critical for the engraftment, adhesion, and migration of the hematopoietic stem and progenitor cells. We proposed three specific aims to test this hypothesis. First, we will determine the functional roles of mDia2 in HSPC engraftment, adhesion, and migration to the bone marrow. Specifically, we will investigate the role of mDia2 in the in vitro and in vivo migration and adhesion of HSPCs and their relationship with the bone marrow niche. We will also use an mDia activator in vivo to explore a pre-clinical model of mDia activation for enhancing stem cell mobilization and engraftment. In these experiments, relatively pure SLAM+ hematopoietic stem cell population will also be tested. Second, we will determine the mechanisms of mDia2 in regulating HSPC engraftment, adhesion, and migration through a novel mDia2-MAL-SRF-LFA1/Mac1 pathway. Third, based on our preliminary studies that acetylation of mDia2 affects its activity in actin polymerization, we will determine whether and how acetylation or other post-translational modifications of mDia2 influence its functions in HSPC engraftment and migration. The overall goal of this proposed study is to understand the roles of mDia2 in HSPC engraftment, adhesion, and migration and elucidate its mechanisms using in vivo mouse models and transplantation assays. A better understanding of these processes may lead to the identification of novel targets for therapeutic strategies in hematopoietic stem cell transplantation.